CN114344480A

CN114344480A - New application of polypeptide, antibacterial gel material and local drug delivery system

Info

Publication number: CN114344480A
Application number: CN202111443294.1A
Authority: CN
Inventors: 杨莉
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-04-15

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a novel application of antibacterial peptide and hydrogel prepared from the antibacterial peptide and oxidized polysaccharide. The invention aims to solve the technical problems that pathogenic bacteria resistance is easily caused by external antibiotic treatment at present and the treatment effect of common antibiotics on multidrug resistant bacteria is poor. The technical scheme for solving the technical problem is to provide a gel material prepared from antibacterial peptide and oxidized polysaccharide, and meanwhile, antibiotics or other biological treatment active ingredients can be loaded. The hydrogel disclosed by the invention has good capability of inhibiting common pathogenic bacteria or multi-drug resistant bacteria, and can promote the migration of fibroblasts at wounds; the antibiotic can play a good role in combination after being added, can greatly reduce the dosage of the antibiotic when treating multi-drug resistant bacteria, and simultaneously delays the drug resistant process of the bacteria, thereby having good clinical application prospect.

Description

New application of polypeptide, antibacterial gel material and local drug delivery system

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a new application of polypeptide, an antibacterial hydrogel material prepared from antibacterial peptide and polysaccharide, and a local drug delivery system.

Background

Acute skin wounds typically heal within 8 to 12 weeks, but healing and treatment time is prolonged if the wound site is infected, especially with multidrug-resistant (mdr) bacteria that lack effective therapeutic means. The multi-drug resistant bacteria can form a biological film consisting of extracellular polymers on the surface of a wound, so that the multi-drug resistant bacteria generate drug resistance to antibiotics, are difficult to remove by conventional means and are easy to repeatedly infect. The theoretically most desirable way to treat wound infections is by topical administration of antibiotics, since this increases the local drug concentration and avoids systemic drug allergies and other toxic side effects. However, few antibiotics are available for topical use, primarily because topical antibiotics can cause hypersensitivity reactions, repeated infections, bacterial resistance, and the like.

The antibacterial peptide is a natural antibacterial substance with broad-spectrum antibacterial capability, and different from the principle of antibiotics, the cationic antibacterial peptide is mainly contacted with the outer membrane of bacteria through positive charges attached to self cations, so that the membrane structure of the cationic antibacterial peptide is disturbed, and the cationic antibacterial peptide is killed. The antibacterial mechanism makes the bacteria difficult to generate drug resistance, and can be used together with antibiotics to play a certain synergistic effect.

The hydrogel is an ideal trauma dressing researched in recent years, the soft gel structure can protect wounds from mechanical damage, the water absorption swelling property of the hydrogel can absorb exudate at the wounds, and the good biocompatibility enables the hydrogel not to hinder the recovery of wound skin. Meanwhile, the grid structure of the hydrogel can be used for loading other medicines or bioactive components to help wound healing.

Disclosure of Invention

The invention aims to solve the technical problem that drug-resistant bacteria are easy to generate by locally using antibiotics, so that the application range of the antibiotics is narrow.

The technical scheme for solving the technical problems is to provide a gel material prepared from antibacterial peptide and oxidized polysaccharide. The antibacterial gel material is prepared by taking antibacterial peptide and polysaccharide as main raw materials; the amino acid sequence of the antibacterial peptide is VQWRIRVAVIRK (SEQ ID No.4), or is a polypeptide which is subjected to 1,2 or 3 insertion, deletion or substitution mutation on the basis of the sequence. Further, the polysaccharide is oxidized polysaccharide. Further, the oxidized polysaccharide is a polysaccharide in which a part or all of hydroxyl groups are oxidized into aldehyde groups.

Wherein the polysaccharide is rich in ortho hydroxyl. Wherein most or all of the hydroxyl groups oxidized into aldehyde groups are ortho-hydroxyl groups.

Further, the polysaccharide rich in ortho-hydroxyl is as follows: at least one of dextran, pullulan, chitosan, carboxymethyl chitosan, hyaluronic acid, sodium alginate, sodium carboxymethyl cellulose or chondroitin sulfate.

Preferably, the polysaccharide is at least one of dextran, pullulan or sodium methyl cellulose.

More preferably, the polysaccharide has a molecular weight of 1 to 10 ten thousand daltons.

Wherein the mass ratio of the antibacterial peptide to the polysaccharide is as follows: the ratio of polysaccharide to polysaccharide is 0.2-0.8: 1.

Wherein the oxidation degree of the polysaccharide in the gel material is between 30 and 75 percent. The degree of oxidation generally refers to the ratio of the number of moles of sugar units oxidized to the total number of moles of sugar units. The oxidized saccharide units can be calculated by determining the number of aldehyde groups contained in the polysaccharide. Preferably, the polysaccharide has an oxidation degree of between 40% and 65%.

The hydrogel is obtained by directly mixing cationic polypeptide and oxidized polysaccharide solution for crosslinking and curing. Wherein the crosslinking curing can be carried out at room temperature.

Further, the solvent of the above solution is normal saline or phosphate buffer. The pH of the phosphate buffer was 7.4.

Wherein, the gel material is prepared by the following method: the polypeptide and the polysaccharide are self-assembled into the hydrogel material in the solution.

Further, the method specifically comprises the following steps:

a. weighing antibacterial peptide and dissolving in a solvent;

the solvent can be at least one of water, phosphate buffer solution or sodium chloride solution

b. Weighing oxidized polysaccharide and dissolving in a solvent;

c. mixing the solutions obtained in the steps a and b at room temperature, and standing until gel is formed;

or, the preparation method comprises the following steps:

a. weighing cationic polypeptide and oxidized polysaccharide, and mixing uniformly;

b. adding a solvent into the solid mixture in the step a to obtain a solution, and standing until a gel is formed.

Wherein the method further comprises the step of freeze-drying the gel or freezing the gel at the temperature of-80-0 ℃ and then thawing the gel again.

Wherein, the amino acid sequence of the polypeptide in the gel material is any one of the following tables:

amino acid sequence	Sequence numbering
		VQWRIRVCVIRA	SEQ ID No.1
VQWRIRIAVIRA	SEQ ID No.2
		VQLRIRVCVIRR	SEQ ID No.3
VQWRIRVAVIRK	SEQ ID No.4
		VQLRIRVCVIRK	SEQ ID No.5
VQWRIRIAVIRK	SEQ ID No.6
		VQWRIRVCVIRR	SEQ ID No.7
VQWRIRICVIRA	SEQ ID No.8
		VQWRIRVAVIRA	SEQ ID No.9
VQWRIRIAVIRR	SEQ ID No.10
		VQWRIRICVIRR	SEQ ID No.11
VQWRIRVAVIRR	SEQ ID No.12
		VQWRIRICVIRK	SEQ ID No.13
VQWRIRVCVIRK	SEQ ID No.14
		VQLRIRVAVIRR	SEQ ID No.15
VQLRIRVAVIRK	SEQ ID No.16

。

Wherein, the polypeptide in the gel material is subjected to amidation modification at the carbon end or acetylation modification at the nitrogen end.

Furthermore, the carbon end of the polypeptide VQWRIRVAVIRK in the gel material is amidated and modified to be VQWRIRVAVIRK-NH₂。

Further, the above, wherein the polypeptide has the structure:

the invention also provides the application of the gel material in preparing a drug delivery system.

The invention also provides a drug delivery system prepared by loading the gel material with the drug.

Wherein, the drug in the drug delivery system is one or more of micromolecular drugs or protein polypeptide drugs; or the medicine is a nanoparticle loaded with one or more of small molecule medicines or protein polypeptide medicines.

Wherein, the small molecule drug in the drug delivery system is an antibiotic.

Wherein the antibiotic in the drug delivery system is at least one of beta lactam antibiotic, macrolide antibiotic, quinolone antibiotic or sulfonamide antibiotic.

Wherein the antibiotic in the drug delivery system is at least one of ceftazidime, vancomycin, imipenem or meropenem.

Wherein, the protein polypeptide drug in the drug delivery system is at least one of cell growth factor or immunocytochemokine.

Wherein the mass ratio of the antibacterial peptide to the micromolecular drug in the drug delivery system is 0.1-1000: 1 as raw material. Further, the mass ratio of the antibacterial peptide to the small molecule drug is 1-600: 1

Wherein, the drug delivery system in the drug delivery system and the nanoparticles loaded with one or more of small molecule drugs or protein polypeptide drugs are mixed according to the mass ratio: 0.1-10: 1 is prepared by taking the raw materials as raw materials. Further, the drug delivery system and the nanoparticles loaded with one or more of small molecule drugs or protein polypeptide drugs are mixed according to the mass ratio of 1-10: 1 is prepared by taking the raw material as a raw material

Wherein, in the drug delivery system, the antibacterial peptide and the protein polypeptide drug are mixed according to the mass ratio of 0.1-10: 1 is prepared by taking the raw materials as raw materials. Further, the mass ratio of the antibacterial peptide to the protein polypeptide medicament is 1-10: 1.

the drug delivery system is prepared by mixing oxidized polysaccharide solution, delivered drug solution and cationic polypeptide solution, and then crosslinking and curing.

Wherein the drug delivery system is a local drug delivery system.

The invention also provides a medicinal preparation prepared by taking the medicament delivery system as a main component. Furthermore, the dosage form of the pharmaceutical preparation is gel, cream, ointment, film or spray applied to the skin and/or the mucosal surface.

The invention also provides a method for preparing the drug delivery system and the drug preparation

The invention also provides the application of the drug delivery system and the drug preparation in preparing products for treating and/or preventing bacterial or drug-resistant bacterial infection of wounds, diabetic foot ulcers and wound infection, or promoting wound healing or skin repair.

The invention also provides the drug delivery system and the application of the drug preparation in preparing products capable of activating the phosphorylation activity of Akt protein in cells or promoting the migration of fibroblasts. Further, the product is at least one of a medicine, a disinfectant, or a cosmetic.

Meanwhile, another aspect of the invention provides the use of a polypeptide, or a derivative of a polypeptide, or a chemically modified product of a polypeptide, in any one of:

a. preparing a product for promoting skin repair; or promoting skin repair;

b. preparing a product for promoting wound healing; or promoting wound healing;

c. preparing a product for preventing and/or treating diabetic foot ulcers; or preventing and/or treating diabetic foot ulcers;

the amino acid sequence of the polypeptide is VQWRIRVAVIRK, or the polypeptide is subjected to 1,2, 3 or 4 insertion, deletion or substitution mutations on the basis of the sequence.

The invention also provides the use of a polypeptide, or a derivative of a polypeptide, or a chemically modified product of a polypeptide, in any one of:

a. preparing a product capable of activating the phosphorylation activity of Akt protein in cells; or activating Akt protein phosphorylation activity in a cell;

b. preparing a product capable of promoting migration of fibroblasts; or to promote fibroblast migration.

The amino acid sequence of the polypeptide is VQWRIRVAVIRK, or the polypeptide is subjected to 1,2 or 3 insertion, deletion or substitution mutations on the basis of the sequence.

Wherein, the amino acid sequence of the polypeptide in the application is any one of the following tables:

。

Wherein the polypeptide is subjected to amidation modification at the carbon end or acetylation modification at the nitrogen end.

Wherein the product is at least one of medicine, disinfectant or cosmetic.

Wherein the dosage form of the product is gel, cream, ointment, film or spray applied to the skin and/or mucosa surface.

Wherein, the carbon end of the polypeptide VQWRIRVAVIRK is amidated and modified to VQWRIRVAVIRK-NH₂。

Further, the polypeptide structure is:

the invention has the beneficial effects that: the VQWRIRVAVIRK polypeptide used in the invention can be cross-linked with oxidized polysaccharide to prepare antibacterial hydrogel, and the antibacterial hydrogel can be loaded with other drugs. The wound healing promoting agent can effectively treat wound infection and accelerate wound healing, especially can play a good role in treating multi-drug resistant bacteria, and can be used together with other drugs such as antibiotics to play a synergistic role, so that the treatment effect can be improved under the condition of ensuring safety, and the defect that the antibiotic is externally used to easily cause bacterial drug resistance can be overcome. In addition, in addition to excellent bacteriostatic effects, the polypeptides used in the present invention may also function as tissue repair peptides, having the functions of promoting wound healing, tissue repair, etc., which may be achieved by activating AKT protein phosphorylation, as found in one example. In one embodiment of the invention, the ceftazidime is used as a model drug loaded in the hydrogel, and the full-thickness skin injury model of the ordinary and type I diabetic mice for treating ceftazidime drug-resistant bacterial infection obtains good curative effect. The bacteria at the wound can be obviously reduced by carrying out homogenate plate-coating counting on the skin after the administration treatment, the healing process of the skin at the wound can be seen to be faster than that of a control group by observing tissue slices, the healing mode accords with the characteristic of scar-free repair, and the attachment amount of type III collagen and the number of M2 macrophages are higher than that of the control group. Meanwhile, the gel material has the advantages of simple preparation method, less needed raw and auxiliary materials, difficult introduction of components which are easy to be allergic or have poor biocompatibility, good biological safety and good application prospect.

Drawings

FIG. 1 shows the chemical reaction formula of dextran oxidation, DP7 structure and DP7 reaction with oxidized dextran.

FIG. 2 shows the IR spectra of dextran, oxidized dextran, DP7-ODEX hydrogel and CAZ-DP 7-ODEX.

FIG. 3, DP7-ODEX hydrogel grid SEM pictures of varying DP7 content and hydrogel pore size statistics.

FIG. 4 is a graph showing the rheological data of DP7-ODEX hydrogel with different concentrations, a, b and c, which are the results of the measured DP7-ODEX hydrogel with 1% concentration, 2% concentration and 3% concentration of DP 7.

FIG. 5 hydrogel gel formation times at different DP7 concentrations.

FIG. 6 release rates of ceftazidime from CAZ-DP7-ODEX gels at different pH conditions.

FIG. 7 shows the erosion curves of DP7-ODEX hydrogel under different pH conditions.

FIG. 8 shows the comparison of the inhibition abilities of DP7-ODEX, CMCS-ODEX and TAT-ODEX hydrogels to different bacteria, and the strains shown in each group of the histogram are PAO1, K12 and 25923 from left to right.

FIG. 9 shows the inhibitory effect of DP7-ODEX hydrogels at different concentrations of DP7 (1%, 2%, 3%, wt%) on different bacteria, with the partitions in each plate representing 1% DP7 (1%), 2% DP7 (2%), 3% DP7 (3%), and control (NS).

FIG. 10 shows the inhibitory activity of ceftazidime solution, DP7-ODEX hydrogel and CAZ-DP7-ODEX hydrogel against 3 Pseudomonas aeruginosa ceftazidime-resistant bacteria at a ceftazidime concentration of 1/4MIC, and the histograms represent the Control group (Control), the ceftazidime group (CAZ), the DP7-ODEX hydrogel group and the CAZ-DP7-ODEX hydrogel group from left to right in each group.

FIG. 11 shows the inhibition of ceftazidime solution, DP7-ODEX hydrogel and CAZ-DP7-ODEX hydrogel against the biofilms of 3 Pseudomonas aeruginosa ceftazidime-resistant bacteria at a ceftazidime concentration of 1/4MIC, where the histograms represent the Control group (Control), the ceftazidime group (CAZ), the DP7-ODEX hydrogel group and the CAZ-DP7-ODEX hydrogel group from left to right in each group.

Fig. 12. ceftazidime agar gel (agar), DP7-ODEX hydrogel, and CAZ-DP7-ODEX hydrogel in vivo bacteriostasis experiments (sampling count after 3 days of administration) at a ceftazidime concentration of 50ug/ml a. each group of mice was infected with bacteria and 3 days of administration wound photo bar 0.5cm b. tissue homogenate plate after colony growth on LB plate c. statistical results on colony count on petri dish.

FIG. 13 in vivo trauma repair experiment after infection of normal mice with Pseudomonas aeruginosa CQ 941. A is a picture of a back wound of the mouse at different time points, wherein bar is 0.5 cm; B. tracing the shape change of the wound; c is the statistics for wound area, and the histograms represent the Control (Control), Ceftazidime (CAZ), DP7-ODEX hydrogel (DP7), CAZ-DP7-ODEX hydrogel (DP7+ CAZ) from left to right within each group.

Fig. 14 pathological section a of wound tissue sampled at different time points is 3, 7, HE staining of the mouse back wound HE at day 14 and masson staining of wound tissue at day 14 with bar 100 μm; b is the statistics of the area of blue collagen in the Masson staining picture; c was CD31 immunohistochemical staining of wound tissue 7 days after administration, with the arrow indicating partly new vessels of bar 50 μm; d is iNOs (marker M1 macrophages) immunofluorescent staining of wound tissue at day 3 with CD206 (marker M2 macrophages) bar 50 μ M; e is the immunofluorescent staining of wound tissue 3 days after administration labeled with type I collagen and type III collagen bar 100 μm.

FIG. 15 is a trauma repair experiment after infection of type I diabetic mice with Pseudomonas aeruginosa CQ 941. A is the wound picture bar of each group of mice at different time points is 0.2cm B, and the statistical result of the wound area is obtained; the bar charts of B represent, from left to right, the Control group (Control), the ceftazidime group (CAZ), the DP7-ODEX hydrogel group (DP7), and the CAZ-DP7-ODEX hydrogel group (DP7+ CAZ), respectively, within each group.

Fig. 16, promoting effect of DP7 and DP7-ODEX hydrogel on fibroblast migration a. cell scratch test result bar 100 μm b. scratch area statistics in cell scratch test, control group, DP7 group, DP7-ODEX hydrogel group c. transwell cell migration test photograph bar 100 μm d. cell number statistics of migration to the back of transwell e.westernblot result in histogram group, respectively, from left to right.

Detailed Description

The invention creatively designs a hydrogel formed by crosslinking antibacterial peptide and oxidized polysaccharide through Schiff base. Can be used for treating wound infected by multidrug-resistant bacteria. The present inventors have discovered, by chance, that DP-series polypeptides can be self-assembled into gels by chemical cross-linking with polysaccharides oxidized with sodium periodate to form schiff bases. Moreover, the gel is pH sensitive, and under the condition of pH which is slightly acidic (less than or equal to 6), the covalent bond between the polypeptide and the oxidized dextran can be accelerated to be broken, so that the hydrogel is degraded, and the internal drug release is accelerated.

Since the growth of bacteria results in a growth environment with a pH that is acidic, the decomposition of the hydrogel can be accelerated by infected wounds, thereby releasing active polypeptides with bactericidal effects. The invention further provides a technical scheme for filling other medicines into the pH sensitive hydrogel, and particularly can fill antibiotics with synergistic action with the antibacterial peptide, so that local adverse reactions caused by excessive medicine release at one time can be avoided, meanwhile, the sensitivity of the drug-resistant bacteria to the antibiotics can be enhanced by utilizing the antibacterial peptide as a framework component of the hydrogel, the dosage of the antibiotics is greatly reduced, the adverse reactions can be reduced under the condition of not influencing the curative effect, and the drug-resistant process of the bacteria to the antibiotics can be greatly slowed down. Meanwhile, part of the antibacterial peptide can be used as a wound repair peptide besides the antibacterial capability, and the wound tissue healing is promoted by the modes of promoting fibroblast migration, regulating wound inflammatory reaction and the like.

The oxidized polysaccharide used in the invention is prepared by oxidation reaction of sodium periodate and/or hydrogen peroxide and polysaccharide rich in ortho-hydroxyl.

Wherein the polysaccharide rich in ortho hydroxyl groups can be dextran, pullulan, chitosan, carboxymethyl chitosan, hyaluronic acid, sodium alginate, sodium carboxymethyl cellulose, chondroitin sulfate, etc.

A typical polysaccharide oxidation reaction is carried out by mixing sodium periodate and the ortho hydroxyl-rich polysaccharide according to a mass ratio of 0.3-1: 1, reacting in pure water at room temperature for 4-6 hours, adding excessive glycol to terminate the reaction after the reaction is finished, removing impurities through dialysis, and obtaining a product by using a freeze drying or rotary evaporation mode.

The antibacterial peptide used in the invention is mainly DP series polypeptide, especially DP7 polypeptide, which can be directly self-assembled into gel with the oxidized polysaccharide in solution, no other auxiliary reagent or other reaction step is needed, and the formed gel has pH sensitivity. The reason for this specificity may be that when DP7 is dissolved in an aqueous solution with a certain ionic strength (such as physiological saline, phosphate buffered saline or PBS), the free amino groups can be sufficiently exposed to react with the aldehyde groups on the oxidized polysaccharide spontaneously at room temperature to form pH-sensitive schiff base (C ═ N).

The gel material provided by the invention can be used for preparing a drug delivery system. The common preparation method is to mix the oxidized polysaccharide solution, the delivered drug solution and the cationic polypeptide solution, and then cross-link and solidify to obtain the hydrogel material. The prepared gel material can be further subjected to freeze drying treatment or frozen at the temperature of-80-0 ℃ and then thawed again. Hydrogels that are frozen and then thawed again may have higher strength.

Meanwhile, the DP7 series of polypeptides can also play a role in tissue repair peptides, have the functions of promoting wound healing, tissue repair, promoting fibroblast migration and the like, and are found to achieve the effects mainly through a mode of activating AKT protein phosphorylation through experiments. In particular, the present inventors have found that DP7-ODEX hydrogel has an unexpected effect of promoting wound healing and skin repair in addition to a good effect of treating wound infection, and have investigated the reasons for this phenomenon. Literature research and experiments show that the glucan has limited promotion effect on wound healing and tissue repair, and the excellent healing effect and scar-free repair effect cannot be achieved by using the glucan alone.

Experiments show that DP7 and DP7-ODEX hydrogel have strong fibroblast migration promoting effect, and fibroblast migration and proliferation are proved to play a crucial role in accelerating wound healing. DP7 polypeptide is also found to activate AKT protein phosphorylation (p-AKT) in fibroblasts through Westernblot experiments, and previous researches report that the p-AKT protein has obvious promotion effect on migration and proliferation of fibroblasts. Early wound healing is susceptible to a variety of factors (e.g., in addition to migration and proliferation of fibroblasts, bacterial infection or tissue damage is also susceptible to release of proinflammatory factors resulting in inflammation) and thus becomes the most rate-limiting step in wound healing. The DP7 polypeptide has the functions of promoting fibroblast migration, promoting wound healing and skin repair, and has good antibacterial effect, so that the wound can quickly cross the stage, and the time required by the whole process of wound healing and skin repair can be greatly shortened.

The present invention is further illustrated by the following detailed description of examples.

EXAMPLES materials and reagents

Antibacterial peptide DP7 (amidation modification of carbon end to VQWRIRVAVIRK-NH)₂) Dextran (molecular weight 50kDa) (limited Shanghai-derived leaf Biotechnology)Sca), sodium periodate (Shanghai Allan Biotechnology Co., Ltd.), ethylene glycol (Shanghai Allan Biotechnology Co., Ltd.), ceftazidime (Dalian Meiren Biotechnology Co., Ltd.).

The bacteria used were: staphylococcus aureus (Staphylococcus aureus) ATCC 25923(ATCC, American type culture collection), Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO, Escherichia coli (Escherichia coli) K12, Pseudomonas aeruginosa clinical isolate PAO1 CQ184, CQ884, CQ926, CQ941 (chongqing hospital).

Examples preparation and characterization of dextran monoxide (ODEX)

0.8552g of sodium periodate was dissolved in 8ml of ultrapure water, and 1g of dextran was dissolved in 10ml of ultrapure water. Sodium periodate was slowly added dropwise into the dextran solution with stirring. The mixture was then stirred at room temperature for 4h in the dark. After 4h the reaction was completed 5ml of ethylene glycol was added and stirred for 5 minutes to neutralize the excess sodium periodate. The reaction solution was then filled into dialysis bags (molecular weight cut-off 14kDa) and dialyzed in ultrapure water for 48 h. And after the dialysis is finished, obtaining oxidized dextran by using a freeze drying method. Structural characterization of oxidized dextran was obtained by fourier transform infrared spectroscopy (FTIR). The degree of oxidation is determined by colorimetric titration with hydroxylamine hydrochloride.

By FTIR results, it can be seen that the peak length is 1731cm^-1The shock absorption peak at the carbonyl group of oxidized dextran compared to before oxidation indicates that dextran was successfully oxidized. The results in FIG. 2 also show the O-H vibration peak (3000-3700 cm)^-1) C-H vibration peak (2900-3000 cm)^-1) And the vibrational peak of the alpha-1, 6-glycosidic bond (1015 cm)^-1)。

The degree of oxidation of the oxidized dextran was then calculated using hydroxylamine hydrochloride colorimetry. The oxidation degree of glucan is obtained by first measuring the number of aldehyde groups contained in oxidized glucan, calculating the number of oxidized glucose units, and dividing the number of moles of the oxidized glucose units by the number of moles of the total glucose units. Specifically, 10.85g of hydroxylamine hydrochloride was dissolved in 25ml of ultrapure water, and 0.7% (w/v) of methyl orange was added thereto, and the pH was further adjusted to 4.0. Then, 100mg of oxidized dextran was dissolved in 25ml of hydroxylamine hydrochloride solution and stirred at room temperature for 2 hours. This was then titrated with 0.1N sodium hydroxide solution until the solution turned yellow from orange. The pH value at the end of titration was 4.3 which was close to the theoretical color change point of methyl orange of 4.4, the volume of sodium hydroxide consumed was 8ml, and the oxidation degree of the oxidized dextran obtained by the following calculation was 62%.

The degree of oxidation is calculated as:

titration reaction formula of hydroxylamine hydrochloride

Oxidized dextran- (CHO)_n+nH₂Dextran oxide- (CH-N-OH) with N-OH & HCl_n+H₂O+nHCl

HCl+N_aOH＝N_aCl+H₂O

EXAMPLE II preparation and characterization of hydrogels

1. Preparation of DP7-ODEX hydrogel

An appropriate amount of antimicrobial peptide DP7(VQWRIRVAVIRK) powder was weighed out and dissolved in PBS to form a 6% (wt%), solution containing 6. mu.g DP7 in 100. mu.l water (multiplied by a density of about 100. mu.g), and an appropriate amount of oxidized dextran was weighed out and dissolved in PBS to form a 10% (wt%) solution. Equal volumes of DP7 solution and oxidized dextran solution were mixed at room temperature and left to stand for 5 minutes to crosslink and solidify, thus obtaining DP7-ODEX hydrogel (final concentration DP 73%, ODEX 5%).

2. Preparation of Ceftazidime (CAZ) -loaded hydrogel (CAZ-DP7-ODEX)

Dissolving a proper amount of CAZ by using PBS, adding DP7 into a CAZ solution to prepare a solution containing 6% (wt%) DP7 of ceftazidime, mixing the DP7 solution containing ceftazidime with an ODEX solution (10% (wt%) in an equal volume, and standing at room temperature for 5 minutes to obtain the CAZ-DP7-ODEX hydrogel.

3. Characterization of DP7-ODEX gel and CAZ-DP7-ODEX gel

The structure of the prepared DP7-ODEX hydrogel is observed by a scanning electron microscope, and the specific method is that the prepared hydrogel is dehydrated by a freeze drying method, and then the cross section of the hydrogel is observed by the freeze electron microscope. Referring to FIG. 3, it can be seen that the pore size of the 1% (wt%) concentration DP7 hydrogel is more elongated due to the collapse of the hydrogel strength slightly lower than the other two. 1% concentration hydrogel pore size was about 20 μm; the pore size of the 2% and 3% concentration hydrogels was slightly around 50 μm compared to 1%.

FTIR data for DP7-ODEX and CAZ-DP7-ODEX were obtained by using lyophilized samples of hydrogels. As a result, it was found that ODEX was 1731cm after crosslinking into hydrogel^-1The shock peak at (a) is reduced. This is due to the Schiff base chemical crosslinking reaction of DP7 with the aldehyde group of ODEX.

When the preparation method is used, the initial concentration of DP7 can be adjusted between 2% and 6% (namely the final concentration is 1% -3% by weight, and the concentrations in the drawing are all final concentrations), the solution viscosity is too high when the concentration of DP7 is more than 8% (initial concentration), hydrogel is not easy to prepare, the viscosity is too low when the concentration is less than 2%, and the strength is too small to be gelatinized after being crosslinked with ODEX. The final concentration can be increased by directly mixing the two powders and then adding water.

The concentration of CAZ varied from experiment to experiment, and the concentration used in the in vivo experiment was 50 μ g/ml, i.e. 50 μ g ceftazidime was contained in 1ml hydrogel. In vitro bacteriostasis experiments, ceftazidime is administrated 1/4 of test bacteria with Minimum Inhibitory Concentration (MIC) to ceftazidime, for example, PAO1 CQ941 has MIC of 64 mu g/ml to ceftazidime resistant bacteria, when 50 mu l of CAZ-DP7-ODEX hydrogel is added into 1ml of culture medium in experiments, the adding amount of CAZ is 16 mu g, namely 16 mu g of ceftazidime is added into 50 mu l of hydrogel into 1ml of culture medium.

And (4) testing rheological properties. The storage modulus (G ') and the loss modulus (G') were measured as a function of time at 37 ℃ with a torque of 1.571 μ Nm, a test angle of 6.28rad/s, and a test time of 10 min. Rheometer results in fig. 4, the storage modulus of the hydrogels increased with increasing DP7 concentration, and the G' measured for 3 DP7 concentrations of hydrogels were 42Pa, 406Pa, and 420Pa, respectively.

And (5) measuring the gelling time. The gel forming time of the hydrogel is measured by an inversion method, namely the hydrogel is prepared at the bottom of a penicillin bottle, and the crosslinking time of the hydrogel is measured by taking the fact that the hydrogel does not flow downwards in an inverted small bottle as a standard. The gel formation times of DP7-ODEX hydrogels (DEX 5%) with a final concentration of 1%, 2%, 3% of DP7 were determined by inversion, respectively. As shown in FIG. 5, the DP7 concentration was varied from 1% to 3%, and the gelling times were 311s, 196s, and 171s, respectively.

EXAMPLE III determination of erosion Rate of DP7-ODEX hydrogels

The erosion rates of the hydrogels were determined in two media, pH5.0 and pH7.4, respectively, since DP7 was cross-linked with ODEX via schiff base, and thus DP7-ODEX hydrogel may have pH sensitive properties and may accelerate degradation under acidic conditions.

Mu.l of DP7-ODEX gel (DP 73%, ODEX 5%, wt%) were prepared in the bottom of 1.5ml of each EP tube, and 1ml of buffer salt solution pH5.0 or pH7.4 was added to the tubes, and incubated in a shaker (220rpm/min) at 37 ℃ and the release medium was removed at the specified time points, together with EP, to determine the weight, whereby the mass of the remaining hydrogel was calculated as a function of time (FIG. 7). The weight change of the hydrogel in the first 12h two media is not obvious, the hydrogel in the medium with the pH value of 5.0 starts to degrade after 12h, and only about 30 percent of the weight remains after 72 h.

Example release Rate assay of Tetraceftazidime from CAZ-DP7-ODEX gel

Release assay was performed in two media, pH5.0 and pH7.4, respectively, and 200. mu.l of CAZ-DP7-ODEX gel (DP 73%, ODEX 5%, wt%) was prepared in the bottom of 1.5ml of EP tube, and 1ml of buffer salt solution, pH5.0 or pH7.4, respectively, was added thereto, and incubated in a shaker (220rpm/min) at 37 ℃ to extract 200. mu.l of release medium at a prescribed time point and supplement 200. mu.l of the corresponding medium, and the cumulative amount of released CAZ was determined by HPLC after sampling. From fig. 6 it can be seen that ceftazidime is released at a higher rate in acidic medium, nearly 70% of the CAZ is released in medium with ph5.0 at three days, while only about 40% of the CAZ is released in medium with ph 7.4.

EXAMPLE V in vitro bacteriostasis test

1. Comparison of the bacteriostatic ability of different compositions of ODEX hydrogels

DP7-ODEX hydrogel (DP 73%, ODEX 5%, wt%), carboxymethyl chitosan (CMCS) hydrogel CMCS-ODEX (0.5% CMCS, wt%), TAT peptide (GRKKRRQRRRPQ) hydrogel TAT-ODEX (3% TAT, wt%) were prepared separately and 50ul each was placed on the bottom of a 96-well plate. Then taking bacterial liquid of three strains PAO1,25923 and K12, adjusting the concentration to 10 by LB culture medium⁵CFU/mL, adding 200ul of bacterial liquid into each hole of a 96-well plate, and then standing and incubating for 16h at 37 ℃. After the incubation is finished, the bacterium solution is sucked out and placed in a new 96-well plate, and the absorbance is measured at 600 nm. (FIG. 8.)

DP7-ODEX is prepared by preparing 6% (wt%) DP7 PBS, mixing with equal volume of ODEX (10% (wt%) PBS, and standing at room temperature for 3 min.

The CMCS-ODEX is prepared by preparing 1% (wt%) PBS solution of CMCS, mixing with equal volume of 10% (wt%) PBS solution of ODEX, and standing at room temperature for 3 min.

TAT-ODEX is prepared by preparing 6% (wt%) PBS solution of TAT peptide, mixing equal volume of ODEX (10% (wt%) PBS solution with it, adding 1 μ l sodium hydroxide solution (1mM) into the mixture, adjusting to alkalinity, and standing at room temperature for 3 min. TAT peptide can be self-assembled into gel with ODEX, but the solution needs to be adjusted to be alkaline, and the formed hydrogel has poor uniformity.

As shown in FIG. 8, only the DP7-ODEX group could completely inhibit the bacteria (the absorbance is less than 10% of the control group), and the inhibitory effects of CMCS-ODEX and TAT-ODEX were weak, so that the DP7-ODEX hydrogel had a stronger inhibitory effect than the hydrogel prepared from other antibacterial materials.

2. Comparison of the bacteriostatic ability of DP7-ODEX hydrogels at different DP7 concentrations

50ul of DP7-ODEX hydrogel with the concentration of 1%, 2% and 3% (DP7, wt%) is prepared at the bottom of a 96-well plate, a control group is 50ul of common agar (agar), and 10ul of PAO1 and K12,25923 bacteria liquid (10 ul of PAO1 and K12,25923 bacteria liquid) are respectively dripped on the surface of the hydrogel⁵CFU/mL, LB medium), left to stand at 37 ℃ for 4h, then the surface of the hydrogel was rinsed with 200ul PBS, then 20ul of rinsed PBS was dropped onto the surface of LB agar solid medium, left to stand at 37 ℃ for 16h, and then taken out for observation (FIG. 9). From lighting the culture dishAs can be seen from the sheet, 3 kinds of bacteria except the control group could not grow on the surface of LB agar solid medium after co-incubation with DP7-ODEX hydrogel at 3 concentrations, demonstrating that direct contact with DP7-ODEX hydrogel could kill these bacteria.

3. In vitro inhibition experiment on drug-resistant bacteria

Separately, 50. mu.l each of DP7-ODEX hydrogel (3% DP7, wt%), ceftazidime solution, CAZ-DP7-ODEX hydrogel (3% DP7, wt%) was placed on the bottom of a 96-well plate. Then, clinical drug-resistant strains (all drug-resistant to ceftazidime) CQ184, CQ884 and CQ941 of PAO1 are taken to adjust the concentration of the bacteria liquid to 10 by using LB culture medium⁵CFU/mL, 200. mu.l was added to a hydrogel-plated 96-well plate. In the control group, the bacteria solution is added without drugs, and the final concentration of ceftazidime added in the ceftazidime solution group and the CAZ-DP7-ODEX hydrogel group is 1/4 of the MIC (minimum inhibitory concentration) of the corresponding drug-resistant bacteria to ceftazidime. Then incubated at 37 ℃ for 16 h. After incubation, the aspirated bacteria was added to a new 96-well plate and absorbance was measured at 600nm, and the results are shown in FIG. 10.

The result shows that the inhibition ability of ceftazidime to drug-resistant strains is weak at 1/4MIC concentration, although DP7-ODEX hydrogel shows an inhibition effect when used alone, the inhibition effect is not enough to completely inhibit bacteria, and after the ceftazidime is loaded into DP7-ODEX hydrogel and then administered, all 3 clinical drug-resistant strains are completely inhibited (the absorbance is less than 10% of a control group), which indicates that DP7-ODEX can play an inhibition effect on drug-resistant bacteria by itself, the integral inhibition ability can be greatly improved after the ceftazidime is used, and the required antibiotic concentration is obviously lower than MIC.

TABLE 1 minimum inhibitory concentrations of DP7 and ceftazidime against clinically drug resistant bacteria

4. Capability of inhibiting biological membrane of drug-resistant bacteria in vitro

Separately, 50. mu.l each of DP7-ODEX hydrogel (3% DP7, wt%), ceftazidime solution, CAZ-DP7-ODEX hydrogel (3% DP7, wt%) was placed in a 24-well plateAnd (4) bottom. Then taking PAO1 clinical drug-resistant strains (all resistant to ceftazidime) CQ184, CQ884 and CQ941 to adjust the concentration of the bacteria liquid to 10 by using LB culture medium⁸CFU/mL, 1mL was added to hydrogel-plated 24-well plates. In the control group, the bacteria solution is added without drugs, and the final concentration of ceftazidime added in the ceftazidime solution group and the CAZ-DP7-ODEX hydrogel group is 1/4 of the MIC (minimum inhibitory concentration) of the corresponding drug-resistant bacteria to ceftazidime. Then incubated at 37 ℃ for 48 h. After the incubation is finished, the culture solution is poured back and washed by PBS, 1ml of crystal violet (0.1 percent, v/v) is added for dyeing for 15min after the moisture in the pore plate is dried, then the crystal violet is washed by PBS and dried, the dyed biomembrane is eluted by 200ul of methanol, and then the methanol dissolved with the crystal violet is added into a new 96 pore plate to measure the absorbance at 590 nm.

The results are shown in FIG. 11. The results show that the influence of the independent use of ceftazidime (1/4MIC) or DP7-ODEX hydrogel on the biofilm formation capability of 3 clinical drug-resistant bacteria is small, but the combined use of the ceftazidime and DP7-ODEX hydrogel can greatly reduce the yield of the biofilm of the clinical drug-resistant bacteria.

EXAMPLE VI in vivo bacteriostasis test

C57 female mice 6 to 8 weeks old were anesthetized by intraperitoneal injection of 10% (v/v) chloral hydrate at a dose of 40. mu.L/10 g, and then the skin on the back was excised, to make a circular wound 8mm in diameter. Then, 10. mu.l of CQ941 bacterial solution (resuspended in physiological saline, 10. mu.l) was added dropwise⁷CFU/ml) on the wound surface, and then different formulations were given for treatment. The therapeutic preparations were DP7-ODEX hydrogel (3% DP7, wt%), agar gel containing ceftazidime (agar, ceftazidime concentration 50. mu.g/ml), CAZ-DP7-ODEX hydrogel (3% DP7, wt%, ceftazidime concentration 50. mu.g/ml) each 50. mu.l, and 50. mu.l of physiological saline was administered to the control group, respectively. Taking down the wound skin after 3 days of administration, homogenizing, taking homogenate diluent, coating a plate, counting and counting the number of wound colonies.

The results are shown in FIG. 12. The results show that after 3 days of administration treatment, the inhibition capacity of the CAZ and DP7-ODEX hydrogel treatment group on bacteria is not greatly different, but the bacterial load on the wound is obviously reduced after the CAZ and DP7-ODEX hydrogel treatment group are combined, and the DP7-ODEX hydrogel and the antibiotics can play a good role in combined action.

EXAMPLE VII experiment for promoting healing of wounds infected with bacteria in vivo

C57 female mice 6 to 8 weeks old were anesthetized by intraperitoneal injection of 10% (v/v) chloral hydrate at a dose of 40. mu.L/10 g, and then the skin on the back was excised, to make a circular wound 8mm in diameter. Then, 10. mu.l of CQ941 bacterial solution (resuspended in physiological saline, 10. mu.l) was added dropwise⁷CFU/ml) on the wound surface, and then different formulations were given for treatment. The therapeutic preparations were DP7-ODEX hydrogel (3% DP7, wt%), agar gel containing ceftazidime (agar, ceftazidime concentration 50. mu.g/ml), CAZ-DP7-ODEX hydrogel (3% DP7, wt%, ceftazidime concentration 50. mu.g/ml) each 50. mu.l, and 50. mu.l of physiological saline was administered to the control group, respectively. The medication was changed every 3 days while the wound size was recorded by taking pictures on

days

0, 3, 7, and 14 (fig. 13). The wound tissue was removed simultaneously and the structure was observed in a pathological section (FIG. 14). HE staining, Masson staining, CD31 immunohistochemical staining, iNOs/CD206 immunofluorescent staining, and type I/III collagen immunofluorescent staining were performed, respectively.

As can be seen by comparing the sizes of wounds on the back of mice, the healing rate of the mice treated with CAZ-DP7-ODEX hydrogel was significantly better than that of the other experimental groups at day 14. It can be seen from the HE staining that the regeneration of the skin appendages such as the pilosebaceous glands was clearly observed on

days

7 and 14 in the DP7-ODEX and CAZ-DP 7-ODEX-treated mice (FIG. 14A). Better collagen adhesion was seen in the skin of hydrogel treated mice on day 14 as seen by Masson staining, and the area of collagen adhesion is statistically shown in figure 14B (figure 14B). Labeling of neovasculature with CD31 revealed more neovasculature in the wound tissue of the mice in the hydrogel treated group (fig. 14C). iNOs can mark M1 type macrophages, which are pro-inflammatory macrophages, whereas CD206 marked M2 type macrophages are anti-inflammatory macrophages, and the excess of M2 type macrophages over M1 type indicates a weaker inflammatory response in the wound, favoring wound healing and reduction of scar tissue (fig. 14D). The proportion of type III collagen to type I collagen is also closely related to scar-free repair, and the amount of type III collagen deposited after hydrogel treatment is significantly higher than type I collagen, which also indicates that the wound healing status after treatment is more ideal and is close to the characteristics of scar-free repair (fig. 14E).

EXAMPLE VII experiment for promoting healing of diabetic mice infected wound

C57 female mice aged 6 to 8 weeks were fasted overnight, followed by intraperitoneal injection of streptozotocin (STZ, 10% w/w, dissolved in citrate buffer salt pH 4.5) at a dose of 120mg/kg, and after 7 days, the blood glucose level of the mice was measured, and those with a blood glucose level of 11.1mmol/L or more were considered as diabetic mice. Diabetic mice were anesthetized by intraperitoneal injection of 10% (v/v) chloral hydrate at a dose of 40. mu.L/10 g, and then the skin on the back was excised, to thereby prepare a circular wound having a diameter of 8 mm. Then, 10ul of CQ941 bacterial solution (resuspended in physiological saline, 10 ul) was added dropwise⁷CFU/ml) on the wound surface, and then different formulations were given for treatment. The therapeutic preparations were DP7-ODEX hydrogel (3% DP7, wt%), agar gel containing ceftazidime (agar, ceftazidime concentration 50ug/ml), CAZ-DP7-ODEX hydrogel (3% DP7, wt%, ceftazidime concentration 50ug/ml) each 50. mu.l, and 50. mu.l of physiological saline was administered to the control group, respectively. The medication was changed every 3 days while the wound size was recorded by taking pictures on

days

0, 3, 7, and 14. FIG. 15 shows that the wound area of the mice treated with the CAZ-DP7-ODEX hydrogel is the smallest at day 14 by comparing the sizes of the wounds on the backs of the mice, which indicates that the treatment effect of the CAZ-DP7-ODEX hydrogel is superior to that of other experimental groups, and also proves that the DP7-ODEX hydrogel and the CAZ-DP7-ODEX hydrogel have good treatment effects in a diabetes trauma infection model.

Example eight experiments to promote cell migration

In the previous experiments, after the DP7-ODEX hydrogel is unexpectedly found to have good effects of promoting skin repair and wound healing, further experiments are carried out for researching the reason that the DP7-ODEX hydrogel promotes skin repair and wound healing. The migration capability of the fibroblasts is directly related to whether the early wound healing condition is ideal, and the early wound healing is easily the rate-limiting step in the whole wound healing process; that is, the migration of fibroblasts is directly related to the speed and repair effect of wound healing and skin repair. Therefore, two experiments are designed, one is to respectively examine whether DP7 and DP7-ODEX hydrogel respectively have the function of promoting fibroblast migration by utilizing a cell scratch experiment and a transwell migration experiment, and the other is to specifically explore the influence of DP7 on the protein expression level of fibroblasts by utilizing a Westernblot experiment.

Experimental results show that the DP7 and DP7-ODEX hydrogel has the function of promoting the migration of fibroblasts. It has also been found that DP7 polypeptide can activate the phosphorylation of AKT protein in fibroblasts, and the phosphorylated AKT protein has been shown to promote the migration and proliferation of fibroblasts in many reports. It follows that the DP7 polypeptide has been shown to have a function in promoting skin repair and wound healing mainly due to the activation of the phosphorylation process of the fibroblast AKT protein by the DP7 polypeptide.

1. Cell scratch test

NIH 3T3 cells were seeded in 6-well plates (3X 10 cells per well)⁵Cells), cells were incubated with DP7 solution (10 μ g/ml) or DP7-ODEX hydrogel (DP 73%, ODEX 5%, wt%, 50ul) for 24 hours after cell attachment. Thereafter, the cells were washed with PBS, the DP7 solution or DP7-ODEX hydrogel was removed, the 6-well plate was scratched with a 10ul gun tip, the exfoliated cells were washed with PBS, and PBS was replaced with DMEM medium containing 1% (v/v) fetal bovine serum. And the scratch change was recorded by taking pictures at time points of 0, 12 and 24 hours and the scratch area was recorded using ImageJ software. Control cells were not treated. It can be seen from FIG. 16A that the migration rate of NIH 3T3 cells was higher after DP7 and DP7-ODEX hydrogel treatment than that of the control group, and the healing rate of the scratch was faster, and it can be concluded from the statistical results in FIG. 16B that the healing rate of the scratch was much higher after DP7 and DP7-ODEX hydrogel treatment than that of the control group.

2. Transwell migration experiment

NIH 3T3 cells were seeded in 6-well plates (3X 10 cells per well)⁵Cells), cells were incubated with DP7 solution (10ug/ml) or DP7-ODEX hydrogel (DP 73%, ODEX 5%, wt%, 50ul) for 24 hours after cell attachment. The cells were then digested, loaded into the upper transwell chamber (6-well plates, 10 ten thousand cells per well), then the lower chamber was loaded with DMEM medium containing 1% (v/v) fetal bovine serum and incubated for 6h at 37 deg.CAfter time, cells in the upper chamber were erased using a cotton ball, and the cells migrated to the back were fixed with 4% paraformaldehyde for 5 minutes, stained with 0.1% (v/v) crystal violet for 15min, and finally, unstained crystal violet was washed off with PBS, and the cells migrated to the back were observed using an inverted microscope (Nikon, FHEIPSE Ti, Japan). From FIG. 16C, it can be seen that the amount of cells migrating to the reverse side of the transwell was greater in the same time period in the NIH 3T3 cells after the treatment with DP7 and DP7-ODEX hydrogel, and this result can be seen from the statistics of the cell number in FIG. 16D. The proliferation and migration ability of fibroblasts are closely related to the wound healing speed, and the promotion of the migration ability of the fibroblasts by DP7 and DP7-ODEX hydrogel indicates that the application of the hydrogel to the trauma treatment is helpful for the healing of wound skin.

3. Westernblot experiment explores the mechanism of DP7 for promoting fibroblast migration

NIH 3T3 cells were seeded in 6-well plates (3X 10 cells per well)⁵Cells), cells were incubated with DP7 solution (10ug/ml) or DP7-ODEX hydrogel (DP 73%, ODEX 5%, wt%, 50ul) for 24 hours after cell attachment. Then extracting protein by using RIPA lysate, and carrying out Westernblot experiment verification. The Westernblot band of fig. 16E shows that the expression level of Akt protein was comparable between groups 3, but the expression level of phosphorylated p-Akt protein was elevated in NIH 3T3 cells treated with DP7 and DP7-ODEX hydrogel, and the Akt protein was activated after phosphorylation to function, which was related to cell migration and proliferation, so it can be concluded that DP7 and DP7-ODEX hydrogel promote fibroblast migration mainly due to DP7 activating the phosphorylation pathway of Akt protein in cells.

Sequence listing

<110> Sichuan university

<160> 16

<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Val Cys Val Ile Arg Ala

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Ile Ala Val Ile Arg Ala

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Leu Arg Ile Arg Val Cys Val Ile Arg Arg

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Val Gln Trp Arg Ile Arg Val Ala Val Ile Arg Lys

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Leu Arg Ile Arg Val Cys Val Ile Arg Lys

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Ile Ala Val Ile Arg Lys

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Val Cys Val Ile Arg Arg

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Val Gln Trp Arg Ile Arg Ile Cys Val Ile Arg Ala

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Val Ala Val Ile Arg Ala

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Ile Ala Val Ile Arg Arg

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Ile Cys Val Ile Arg Arg

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Val Ala Val Ile Arg Arg

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Ile Cys Val Ile Arg Lys

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Val Cys Val Ile Arg Lys

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Leu Arg Ile Arg Val Ala Val Ile Arg Arg

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<213> Artificial Sequence (Artificial Sequence)

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Val Gln Leu Arg Ile Arg Val Ala Val Ile Arg Lys

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Claims

1. The antibacterial gel material is characterized by being prepared from antibacterial peptide and polysaccharide serving as main raw materials; the amino acid sequence of the antibacterial peptide is VQWRIRVAVIRK (SEQ ID No. 4); or a polypeptide mutated by 1,2 or 3 insertions, deletions or substitutions on the basis of its sequence.

2. The gel material of claim 1, wherein: the polysaccharide is oxidized polysaccharide.

3. The gel material according to any one of claims 1 or 2, wherein: the polysaccharide used is a polysaccharide rich in vicinal hydroxyl groups.

4. A gel material according to any one of claims 1 to 3, wherein: further, the polysaccharide rich in ortho-hydroxyl is as follows: at least one of dextran, pullulan, chitosan, carboxymethyl chitosan, hyaluronic acid, sodium alginate, sodium carboxymethyl cellulose or chondroitin sulfate.

5. A gel material according to any one of claims 1 to 4, wherein: the polysaccharide is at least one of glucan, pullulan or sodium methyl cellulose.

6. A gel material according to any one of claims 1 to 5, wherein: the molecular weight of the polysaccharide is 1-10 ten thousand daltons.

7. A gel material according to any one of claims 1 to 6, wherein: the mass ratio of the antibacterial peptide to the polysaccharide is that the antibacterial peptide is 0.2-0.8: 1.

8. A gel material according to any one of claims 1 to 7, wherein: the oxidation degree of the polysaccharide is 30-75%; preferably, the oxidation degree of the polysaccharide is 40-65%.

9. A gel material according to any one of claims 1 to 8, characterized in that it is prepared by a process comprising: the polypeptide and the polysaccharide are self-assembled into the hydrogel material in the solution.

10. The gel material of claim 9, characterized in that it is made by a process

a. Weighing antibacterial peptide and dissolving in a solvent;

b. Weighing oxidized polysaccharide and dissolving in a solvent;

or, the preparation method comprises the following steps:

11. The gel material of claim 9 or 10, wherein: further comprises the step of freeze-drying the gel or freezing the gel at a temperature of-80 ℃ to 0 ℃ and then thawing the gel again.

12. The gel material of any one of claims 1 to 11, wherein: the amino acid sequence of the polypeptide is any one of the following tables:

。

13. Use according to any one of claims 1 to 12, wherein: the polypeptide is subjected to amidation modification at the carbon end or acetylation modification at the nitrogen end.

14. Gel material according to one of claims 1 to 13, wherein the polypeptide is VQWRIRVAVIRK-NH amidated at the carbon-terminal of VQWRIRVAVIRK₂。

15. Gel material according to any one of claims 1 to 14, characterized in that the antibacterial peptide has the structure:

16. use of a gel material according to any one of claims 1 to 15 in the preparation of a drug delivery system.

17. A drug delivery system prepared by loading a drug into the gel material of any one of claims 1 to 15.

18. The drug delivery system of claim 17, wherein the drug is one or more of a small molecule drug or a protein polypeptide drug; or the medicine is a nanoparticle loaded with one or more of small molecule medicines or protein polypeptide medicines.

19. The drug delivery system of claim 18, wherein said small molecule drug is an antibiotic.

20. A drug delivery system according to claim 19, characterized in that said antibiotic is at least one of a β -lactam antibiotic, a macrolide antibiotic, a quinolone antibiotic or a sulphonamide antibiotic.

21. A drug delivery system according to claim 19, characterized in that said antibiotic is at least one of ceftazidime, vancomycin, imipenem or meropenem.

22. The drug delivery system of claim 18, wherein said protein polypeptide drug is at least one of a cell growth factor or an immunocytochemokine.

23. A drug delivery system according to any one of claims 1 to 22, wherein: the mass ratio of the antibacterial peptide to the micromolecular drug is 0.1-600: 1 as raw material.

24. A drug delivery system according to any one of claims 1 to 23, characterized in that: the mass ratio of the drug delivery system to the nanoparticles loaded with one or more of small molecule drugs or protein polypeptide drugs is 0.1-10: 1 is prepared by taking the raw materials as raw materials.

25. A drug delivery system according to any one of claims 1 to 24, characterized in that: the mass ratio of the antibacterial peptide to the protein polypeptide medicine is 0.1-10: 1 is prepared by taking the raw materials as raw materials.

26. A drug delivery system according to any one of claims 1 to 25, characterized in that: is prepared by mixing oxidized polysaccharide solution, delivered drug solution and cationic polypeptide solution, and then crosslinking and solidifying.

27. A drug delivery system according to any one of claims 1 to 26, characterized in that: the drug delivery system is a topical drug delivery system.

28. A pharmaceutical formulation comprising as a main carrier material a gel material as claimed in any one of claims 1 to 15 or as a main ingredient a drug delivery system as claimed in any one of claims 17 to 27.

29. The pharmaceutical formulation according to claim 28, wherein the pharmaceutical formulation is in the form of a gel, cream, ointment, film or spray for application to the skin and/or mucosal surface.

30. A process for the preparation of a drug delivery system according to any one of claims 17 to 27 or a pharmaceutical formulation according to claim 28 or 29.

31. Use of a drug delivery system according to any one of claims 17 to 27 or a pharmaceutical formulation according to claim 28 or 29 in the manufacture of a product for the treatment and/or prevention of bacterial or drug-resistant infection of a wound, a diabetic foot ulcer, a wound infection, or for promoting wound healing or skin repair.

32. Use of a drug delivery system according to any one of claims 17 to 27 or a pharmaceutical formulation according to claim 28 or 29 for the preparation of a product capable of activating the phosphorylation activity of Akt proteins in cells or a product capable of promoting fibroblast migration.

33. Use according to claim 31 or 32, characterized in that the product is at least one of a pharmaceutical, a disinfectant or a cosmetic product.

34. Use of a polypeptide, or a derivative of a polypeptide, or a chemically modified product of a polypeptide, in any one of:

a. preparing a product for promoting skin repair; or promoting skin repair;

b. preparing a product for promoting wound healing; or promoting wound healing;

c. preparing a product for preventing and/or treating diabetic foot ulcer; or preventing and/or treating diabetic foot ulcer;

35. Use of a polypeptide, or a derivative of a polypeptide, or a chemically modified product of a polypeptide, in any one of:

36. Use according to any one of claims 34 or 35, wherein: the amino acid sequence of the polypeptide is any one of the following tables:

。

37. Use according to any one of claims 34 to 36, wherein: the polypeptide is subjected to amidation modification at the carbon end or acetylation modification at the nitrogen end.

38. Use according to any one of claims 34 to 37, wherein: the product is at least one of medicine, disinfection product or cosmetics.

39. Use according to any one of claims 34 to 38, wherein: the product is in the form of a gel, cream, ointment, film or spray for application to the skin and/or mucosal surface.

40. Use according to any one of claims 34 to 39, wherein: the carbon end of the polypeptide VQWRIRVAVIRK is amidated and modified to VQWRIRVAVIRK-NH₂。

41. The use according to any one of claims 35 to 40, wherein the polypeptide has the structure: